Summary Intrinsically disordered proteins (IDPs) make up more than 30% of eukaryotic proteomes. They carry out vital functions in the cell, such as signaling, transcription and translation, and they regulate and control the cell cycle. Their malfunction leads to some of the most challenging diseases, of growing concern to the US health care system, such as cancer and neurodegeneration. The associated cost of these diseases are some of the highest and fastest growing in the US. IDPs also play a key role in the replication and spreading of viral pathogens. In order to function properly IDPs must (i) bind efficiently to specific binding partners; and (ii) avoid pathological aggregation. A molecular level understanding of how IDP sequences encode for these two processes, and how mutations and stress conditions in cells can affect them, will significantly advance our understanding and ability to treat and prevent such diseases. Major experimental efforts are currently focused on: (i) resolving the structural ensembles of IDPs and the binding mechanism (IDP structure/function); or (ii) resolving the mechanisms of IDP aggregation into IDP aggregation into specific amyloid aggregates (IDP malfunction). Here we propose a radically different, physical chemistry approach in which we study the effect of IDP conformational dynamics on the binding mechanism; and the quantitative relation between IDP phase separation and aggregation. We will do so by using high resolution experimental techniques and methods developed in our lab, along with the multiscale simulations. Because most IDPs bind through coupled folding and binding, their conformational dynamics is expected to greatly affect the binding mechanism. Our approach of incorporating IDP conformational dynamics in binding studies will provide a key missing link to understand IDP functional binding. Our proposed study builds on the recent characterization of the binding mechanism of a group of IDPs, and focuses on studying 1) the effect of conformational dynamics on binding; and 2) the physiological process of liquid phase-separation and its link to pathological aggregation. We will combine different high resolution experimental techniques -including nanosecond laser pump spectroscopy- with molecular simulations to characterize IDP structure and dynamics. Novel methods will be used to quantify liquid phase-separation of IDPs. Results from Aim I: Test our hypothesis by comparing IDP dynamics for different binding scenarios; Aim II: Modulating the binding mechanism by perturbing the sequence and solvent, and Aim III: Quantify the effect of a disease mutation on the conformational dynamics, phase separation and aggregation of FUS_LC; will have direct impact on the molecular understanding IDPs implicated in ovarian and breast cancer, in the replication of paramoxyviruses and in amyotrophic lateral sclerosis and frontotemporal dementia. These results have the potential of transforming our way of viewing coupled folding and binding, and liquid phase separation which are crucial for IDP function.